I NTE RNATIO NA L J ()l lRNA I. OF I ,EI'ROSY Volu me 4 I , Number :1 Prillted ill 1/.- US.A.

Advances In the Microbiology of M. leprae in the

Past Century

Yoshio Yoshie 1

Facts of profound interest, not merely with respcct to the pathogenesis of leprosy, but for the general hi story of bacteriology are cvident in an attempt to trace the segmcnt of history related to investigations in leprosy following the discovery of the lcprosy bacillus to the establishment of its role as the pathogen causative of the dis­ease.

In Europe, thc most widespread epidem­ics of leprosy occurred about the thirteenth and fomteenth centuries, followed by a gradual decline. The disease was drastical­ly diminished by the middle of the nine­tcenth century, with the exception of a few limited areas in Europe. On the other hand, an extensive epidemic of leprosy developed in Norway in about the middle of the nineteenth century.

In 1847, D . C. Danielssen, the then chief phys ician at the leprosy hospital in Bergen, and C. W . Boeck, the then professor of dermatology at Christiania Medical School published from Christiania the book Om Spedalskhed ( A Study of Leprosy) , whi ch represents an epochal achievement of clini­co-pathologic research on leprosy based on their wide experience and profound knowl­edge of the morbid anatomy of the disease, The classification of leprosy into two princi­pal types, the tubercle (nodular ) type and the anesthetic type, promulgated by them, was indeed a foresight that deserves the highest admiration. T Ie contributions made by these great men in the classifica­tion of leprosy into two principal forms are still shedding light on '"he medical science of leprology.

From his early investigative days, Dan­ielssen had recognized the presen'ce of small brown or yellowish, grossly discerni­ble "granular masses" or "brown elements" demonstrable on histopathologic prepara-

1 Yoshio Yoshie, M.D ., Director , National Insti ­tute for Leprosy Research , Higashi-Murayamashi , T okyo, Japan.

tions from lep rosy nodules and had been convinced of their peculiarity to leprosy_ However, perhaps he did not think that the "masses" had any vital bearing on the etiol­ogy of leprosy. In 1859 Danielssen asked thc opinion of R. Virchow, who was visiting him, of the "brown masses." Virchow was not particularly impressed with Daniels­sen's discovery and interpreted the bodies as representing mere clumps of degenerat­ed fat and, therefore, having nothing to do with leprosy. Years later, Virchow altered this initi al view and designated the brown masses as "lepra cells" (8).

The discovery of the leprosy bacillus. In 1868, Gerhard Henrik Armauer H ansen, as a young phys ician, began to attend his duties at the Nursing Home for Lepers No. 1 and subsequently, in the same year, fill ed the post of assistant physician at Lun­gegaarden H ospitaL In 1870, H ansen, hav­ing returned from a twelve month sojourn at Bonn and Vienna to acquire basic knowledge and skill in pathologic anatomy and mi croscopy, pursued investigations on leprosy in an area of western Norway hav­in g a hi gh incidence of the disease, as well as Lungegaarden H ospitaL

H e summarized the results of these in­vestigations in a paper which he submitted in 1873 to the Norweigian Medical Associa­tion in Christiania, the body by which he had been awarded his research scholarship_ This work, which he entitled "A Report to the Norwegian Medical Association in Christiania on a Journey Undertaken with the Backing of the Association to Investigate the Causes of Leprosy," was printed in 1874 as a supplement to NOBsK MAGAZIN FOB LAEGEVIDENSKABEN (The Norwegian Journal of Medical Science ) (sa )_

This paper contains the first description of the causative agent of leprosy : "There are to be found in every leprous tubercle extirpated from a living individual- and I have examined a great number of them-

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362 1 nternationaU ourlUll of Leprosy 1973

sma]], staff-like bodies, much resembling bacteria, lying within the cells; not in all, but in many of them" (36, 13) . Further­more, he emphasized in an indirect yet presumptive fashion, evidence corroborat­ing the concept that leprosy is a chronic communicable disease caused by a specific agent. H ansen, therefore, eminently deserves credit for the conception of the pathogen he demonstrated as being respon­sible for leprosy since at that time the prevailing view was that leprosy was not a contagious' but a humoral or hereditarily constituted disease,

In what year did Hansen first discover the leprosy bacillus? His discovery of the leprosy bacillus was in 1873 according to one article while another claims the year of discovery to be 1874,

In an editorial in this JOUR NAL in 1964, Wade (36) noted that 1874 was certainly the year of publication by H ansen of his diseovery of the leprosy bacillus. Therefore that year is the recognized date of the discovery according to modern practice,

As a comment on this matter, H, P. Lie, who had been Hansen's assistant and suc­ceeded him, wrote that the observations published in 1874 had been made in the previous year and that consequently Han­sen himself had maintained th:1t the discov­ery of the bacillus must be reckoned as from 1873, Vogelsang stated that if we credit the work to the time it was done rather than to the time of publication of the report, then 1873 is the year in which Armauer Hansen discovered the leprosy bacillus,

In his popular book The Fight Against Leprosy ( 1964) , Patrick Feeny gives what purports to be the actual date of the finding that the brown bodies were masses of indi­vidual rods as February 28, 1873, and even notes the name of the patient concerned, Certain other cases in the same period are also cited.

A brief description of the medical and microbiological background of the days of Hansen's discovery is of interest as related to this discovery, Rudolf Virchow pub­lished in 1858 Die Zellularpathologie in ihrer Begriindung auf physiologische und

pathologische Gewebelehre. Diseuses had generally been believed to exist in the Or­gans at the time of Giovanni Battista Mor­gagni ( 1761) , the founder of modern path­ologic anatomy, and to exist in the tissues at the time of Marie Francois Zavier Bichat ( 1771-1802 ) , With further development, the contemporary cellular pathologic con­cept that diseases have cellular bases was shown by Virchow and his school. Mean­while, advances in microscopy not only contributed much to the development of ccllular pathology but resulted in a great deal of progress in the microbiology of pathogenic organisms, Beginning with the microscope of Antony van Leeuwenhoek ( 1632-1723 ) up to the development of apo­chromat objectives with extremely low aberration by Ernst Karl Abbe (1887), pro­digious progress was made in microscopy and, in consequence, bacteriology as a gen­uine branch of science was born late in the nineteenth century, and thereafter made great strides, In the records of the observa­tion of a great variety of things in the natural world, which Leeuwenhoek saw with the microscope he invented, it is said that there is a description of a group of microorganisms which we nowadays should recognize as bacteria , However, no further progress could be made along this line at his time because science in general was not well-developed enough to permit utili­zation of his observation, Thus, Leeuwen­hoek, as the father of microscopy, intro­duced the concept of microbes but did not become the father of bacteriology.

The first development of contemporary bacteriology was brought about, as is wide­ly known, by the two distinguished scien­tists Louis Pasteur ( 1822-1895) and Robert Koch ( 1843-1910 ), They were the first to demonstrate and describe various activities of I~icroorganisms as pathogenic agents, Thus, great strides were not made until after the lapse of more than a century following mere recognition of the occur­rence ,of microbes, 'Vith a profound interest in the phenomenon of fermentation, Pas­teur as a chemist demonstrated through experiments that various forms of fermen­tation are brought about by actions of spe­cific microorganisms. This dazzling sci en-

41, 3 Y oshie: M iC1'Obiology of M. leprae 363

tific achi cvement exerted a grcat influence upon the subsequent dcvelopmcnt of path­ogenic bacteriology. Threc years follow­ing the discovery of the leprosy bacillus by Hansen, Koch succeeded in cultivating the anthrax bacillus in 1876. In 1881 he devised a solid medium which permitted the forma­tion of colonies as pure bacterial cultures, and in 1882 he identified thc tubercle bacil­lus as thc ctiologic agent of tuberculosis.

Koch was a student of the pathologist Jacob Henle ( 1809-1885), who drew up a statement in 1840 of the conditions re­quired to provide acceptable proof of a casual relationship between an infectious agent and a given disease. These conditions undoubtedly represented his own concepts though they cannot be found in his writ­ings. They were succinctly formulated by Koch and are widely known as "Koch's postulates." These postulates came to con­stitutc the recognized basis for establishing an agent as having speci fic etiologic signifi­cance. Their promulgation contributed greatly to the development of pathogenic bacteriology. Bacteriology marked rapid progress on the bas is of methods of labora­tory studies found by Koch, and there followed in rapid succession the identifica­tion of the typhoid bacillus in 1883, the diphtheria bacillus and cholera vibrio in 1884, the tetanus bacillus in 1886, and in increasing tempo, many others.

The time of H ansen's discovery of the leprosy bacillus, about 1873, was the pre­Pasteur and pre-Koch era when the con­cept of p athogenic bacteriology as yet was scarcely established. There was no proof that leprosy was caused by infection with a living germ, nor was the microscope, as then available, completely adequate.

Five years following Hansen's report on the observations of microscopic bacillary objects in leprous tissue, Albert Neisser, a 24 year old microbiologist, visited Norway in 1879 where he made clinical observa­tions of numerous patients at a leprosy hospital and received various leprous tis­sues from H ansen. H e returned to Breslau where, with the aid of Koch, Weigert and Ehrlich, he made detailed microscopic ex­aminations of the specimens with remark­able skill in staining technics. As a result,

Neisscr published "Ubcr die Atiologie dcs Aussatzes" in 1879 in which he stated: "Thcre were revealed everywhere bacilli in large numbers, in all 14 pieces of skin and nodules." Similar bacilli were demonstra­ble, according to him, in the liver, spleen, lymph nodes, cornea and most abundantly in the tes tis (26).

In· 1879 Hansen further published a drawing of Icprosy bacilli as hc observed them under th c microscope. Neisser contin­ued bacteri ologic cxamlnation of additional leprous material which he had collected in 1881 from Spain, Dutch Guyana, Brazil, Rumani a, thc East Indies, Palestinc and other arcas. In all these ti ssues he demon­stratcd the same bacilIi as those scen in the earlier studies, thus confirming that the bacilli were thc ctiologic agent of the dis­ease.

Subsequently, at thct"irst International Leprosy Congrcss held ' n Berlin in 1897, it was gencrally adni tted that Hansen was the discovcrcr of the leprosy bacillus and that this bacillus wa~ the organism causa­tive of leprosy.

Meanwhile, Weigert in 1875, was the first to devise staining of microorganisms with solutions of dyes, and discoveries of new dyes and mordants as well as advances in the technics of staining foll owed. In 1882 Ehrl ich discovered the 'peculiar straining reactions of tubercle bacilli , which are diffi­cult to stain but when once stained with gentian violet and saturated anilin solution in water they resist decolorization by min­eraI acids; hence, the name aci d-fast stain­ing, This peculiarity of Mycobacteriaceae became the principal method of differenti­ating them from other microorganisms. This acid-fast staining technic was subsequently improved and came to be generally desig­nated as the Ziehl-Neels en stain. In 1879 Hansen called the leprosy organism Bacil­lus leprae, but in 1896 the term Mycobac­terium leprae was proposed by Lehmann and Neumann, and this t erm is generally used at present.

Bacterial cytology of M. leprae. In the hundred years since the discovery of the leprosy bacillus great progress has been made in increasing the 'resolving power and magnifying capacity of the light micro-

364 International Journal of Leprosy 1973

scopc, and the advent of thc electron micro­scopc, which made a rapid progress after the introduction by B. Borries and E. Ruska in 1938 of a transmission electron micro­scope, ,heightened these capacities. Addi­tionally, a variety of excellent staining tech­nics have been devised as the result of the increase in and refinement of useful dyes as well as advances in the theoretical aspects of staining. There resulted a rapid progress in the morphologic observation and de­scrip tion of bacteria and the development of kn()wledge of bacterial cytology.

As for the morphology of M. Zeprae, it has been recognized since the beginning of this century that M. Zeprae varies in mor­phology with the phases of leprosy. Numer­ous leprosy bacilli have been noted to un­dergo various transformations during lepra reactions. Thus, diphtheroid , beaded, spore-like and granular forms, as well as poorly acid-fast organisms have been found to appear.

Promin (glucosulfone sodium) was first tried by Faget et al in 1943, in the treat­ment of leprosy (7), with results so gratify­ing as to be called a miracle at Carville. This orginated the antileprotic chemothera­py of the present day. In association with this development, methods for examination of the quantitative and qualitative changes in leprosy bacilli in smears from the skin and nasal mucosa came to assume consider­able clinical importance in the evaluation of therapeutic effects. The Technical Panel on Bacteriology and Immunology of the Eighth International Congress of Leprology in 1963, provided advice for standardiza­tion of these technics calling for : 1) uni­form methods for obtaining samples and preparing smears; 2) a logarithmic or ar­ithmetical scale for expressing the numbers of concentration of bacilli seen; and 3) classification and interpretation of the fol­lowing morphologic features in M. leprae: solid form, which are thought to b e viable; fragmented or disorganized forms, which arc thought to be damaged or dead; coccoid or granular forms, which mayor may not be viable; acid-fast debris, which likely indicates rapid destruction of bacilli within th e recent past.

As a result of comparative observation of

the appearance of dcad and degenerated leprosy bacilli by light and electron micros­copy, evidence was obtained that the non­solid form demonstrable with the Ziehl­Neelsen stain was suggestive of degener­ate and dead organisms. Accordingly, a hypothesis developed that the solidly and uniformly Ziehl-Neelsen stained M. Zeprae were viable whereas those which were non­solid represented nonviable cells (30).

Meanwhile, experimental studies on in­fectivity as related to the average doubling time of solid bacilli of M. Zeprae was pur­sued by the technic of inoculation into foot pads of micc. A closc relationship was shown to exist between the number of solid staining bacilli and the infectiousness of these bacilli for the mouse foot pad (:14). However, there exists a widespread opposition to the concept that all nonsolid M. Zeprae cells are dead, because in prac­tice the judgment of nonsolidness of the baci llus is not alwavs made.

On the occasion of the WHO meeting of investigators from research centers working on M. Zeprae in 1968, the technics for the preparation of smears, fixation and staining were standardized. Ridley's Bacteriological Index (BI) was recommended as standard for quantitization. In addition, it was rec­ommended that the Morphologic Index (MI ) should not be included in routine bacteriological examination because th e re­sults obtained were too variable to be of value. Furthermore, the Expert Committee on Leprosy of the World H ealth Organiza­tion in 1970, reported on the Morphologic Index (solid ratio) as follows: "It is the most convenient laboratory method avail­able for following the therapeutic response of pati ents in short-term clinical trials ." Although considerable evidence has been provided that the Morphological Index re­flects the viability of M. leprae, "because of its limits of sensitivity, it is not a suitable procedure for distinguishing the infectious from the noninfectious patient, even when performed under optimal conditions by highly expericnced investigators."

Since 1948, when Bishop et al (3) first applied electron microscopy to thc study of the morphology of M. Zeprae, this technic has been utilized widely in leprosy research.

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41, 3 Yoshie: Microbiology of M.leprae 365

Studies have been performed on the ultra­structure of the M. leprae cell (4), on ultrastructure of various types of leprosy lesions (=17), cytoplasmic reactions to the leprosy bacillus and intracytoplasmic en­zymes of the host cell (15. ](I). Nevertheless, there is still inadequate experimental evi­dence to clarify the relationship between the phagocytic membrane and the host­mycobacterial interaction. Recently, some clarification has been made as to the inter­relationship of lysosomal substance and in­tracellular bacilli by cytochemical electron microscopic examination of the lepra cell. It seems highly probable that the relation­ship between the structure and the func­tion or physiologic states of M. leprae will be clarified through cytochemical and en­zymatic studies combined with electron mi­croscopy.

Various investigations have pursued studi ~s on the chemical properties of cellu­lar 20mponents of M. leprae since the studies made by Unna and his son in 1910. Nevertheless, present knowledge in this area is still quite sparse. Anderson et al conducted a series of subtle chemical an­alyses on the constitutents of the tubercle bacilli and other mycobacteria and thereby demonstrated the presence of lipoid (phos­pholipid, lipid, wax, bound lipoid) , pro­tein, carbohydrate and pigments (phthio­col, cartinoid) as the components of myco­bacterial cells and designated neutral wax isolated from M. leprae under the name of "leprosin" (2). However, it is not generally accepted that the cultures analyzed were actually those of M. leprae.

While it is evident from a number of studies that M. leprae shares common sero­logically active antigens with M. tubercu­losis and other mycobacteria, it is almost impracticable to conduct antigenic analyses of M. leprae unless sufficiently large amounts of pure M. leprae cells are avail­able. Nevertheless, it was revealed recently by immunoserological studies, immunodif­fusion and immunofluorescence t ests that M. leprae contains a protein antigen showing an entirely different specificity from other species of mycobacteria (1). A recent electron microscopic study (10) re­vealed that M. leprae possesses, like all

other mycobacteria, a superficial network of filaments fundamentally identical with the adjuvant active peptido-glycolipid filaments. The immunological phenomena common to leprosy and tuberculosis are largely dependent upon the biological ac­tivity of the mycobacterial peptido-glyco­lipid and its ability to res ist the action of host enzyme and is thought to be of impor­tance for its fun ctions as an immunological adjuvant (In). Moreover, in recent chemical analyses and electron microscopic studies, the cell wall of M. lepraemurium was found to bear ultrastructural and chemical resemblance to those of other mycobacteria and to contain such components as muco­peptide, arabino-galactan and ester-linked lipid (6. 17) .

Advances in animal transmission of M. leprae. Insomuch as this subject will be dealt with in detail by another contributor, it will be alluded to only briefly. Despite ',farious attempts at M. leprae inoculation into laboratory animals and sometimes into humans by a number of investigators, in­cluding Hansen and Danielssen, no single case of success has been certainly recorded.

In 1960 Shepard inoculated leprosy bacilli isolated from nasal washings and lesions of lepromatous patients into the foot pads of CFW mice which were maintained at a room temperature of 20° C. The bacilli were noted to show local multiplication when the inoculum was diluted appropri­ately. Thus, when 10:1 to 104 bacilli were injected, five to ten months were required to yield a harvest of 105 to 107 bacilli (3.3). Subsequently, the mouse foot pad infection was adopted in laboratories in many coun­tries and, at present, is employed as a reliable screening procedure for drugs for leprosy. Although the mouse foot pad inoc­ulation technic has failed to produce exper­imental leprosy in mice, this distinguished contribution opened the way anew for ex­perimental an imal transmission of the lep­rosy bacillus which had long been looked forward to by leprologists.

Ensuing foot pad studies have disclosed similar results in other rodent species in­cluding rats , hamsters and mystromys, though varying among species to some ex­tent.

366 International Journal of Leprosy 1973

In normal micc the logarithmic phase of multiplication of M. leprae is maintained for six to eight months, followed by a plateau and then a regression phase. The generation time is 12 to 13 days in the logarithmic phase of multiplication. More recently it has been shown that this limited phase of logarithmic multiplication can be extended by reducing the immunologic ca­paci ty of the mice by thymectomy followed by total body irradiation (900 r) before infection, necessitating a small, life-saving injection of syngeneic bone marrow cells ( :19).

Advances in cultivation of M. leprae. In 1882, after a lapse of several years following his discovery of the leprosy bacil­lus, Hansen tried to cultivate the leprosy bacillus. Studies representing an attempt to cultivate the bacillus have been continued by an extremely great number of investiga­tors in the past one hundred years. These elaborate attempts encompassed trials with practically all conceivable methods, such as varying conditions of cultivation (tempera­ture, aerobiasis, anaerobiasis, O~ and COt gas environment, etc.), investigation of composition of culture media including growth promoting substances, special cul­tivation methods such as symbiotic culture with other bacteria, cultivation by the use of special double tube apparatus and dif­fusion chambers, cultivation in embryonated chicken eggs, tissue culture and m'any other technics. Nevertheless, the organism has never been accepted generally as having been cultured with certainty.

Mycobacterium lepraemurium, the agent causative of leprosy in rats and mice with lesions in the skin and lymph nodes that resemble those of human leprosy, was dis­covered in 1903 by Stefansky. Since this organism also is noncultivatable, M. lep­raemurium has been extensively employed as a model in studies of cultivation of M. leprae.

The strains of acid-fast bacilli which have been isolated in attempts to obtain microbial growth from leprous materials are innumerable. All constitute a history full of value despite the failures associated with them. The following is only a brief description of such attempts in accord with

the classification of these cultivated strains by Rogers and Muir ( 1925) (3J).

Strains belonging to diptheroids or Strep­tothrix. Representative of this group is the bacillus isolated by Kedrowsky (1901) with human placental extract-agar, horse serum agar and so forth . Many of the strains in the diptheroid group are similar in many respects. They are labile with respect to their acid-fast properti es, pleomorphic, and grow readily on the ordinary culture media. Their acid-fastness varies with the culture medium; it diminishes in fate-free media and is enhanced in fat-containing media.

Chromogenic acid-fast bacilli. These have been isolated with the greatest fre­quency and most organisms grown and assumed to be M. leprae frequently fall in this category. Their biologic characteristics seem to be entirely comparable to those of th e so-called spontaneously occurring non­pathogel1ic mycobacteria. They are easily grown, their acid-fastness varies from strain to strain, and they are mostly pleomorphic.

Nonchromogenic acid-fast bacilli. Orga­nisms of this group are generally difficult to subculture. Those isolated by Emile-Weil, Karlinski, Marchoux, Twort, Duval and Wellmann, etc. , belong to this group. The organism isolated by Twort from nasal mucus grew only in a Dorset's egg medium containing 1% tubercle bacilli and could not be subcultured. However, the "Dalva" strain which Souza-Araujo (1952) isolated in Lowenstein's medium from nasal mucus was subcultivable.

A11(lerobic bacilli. Miscellaneous. Among the numerous cultivation attempts that have been made in the pas.t, there have been several experimental findings report­ed to be convincing as probable true growth of M. leprae though only to a microscopically demonstrable extent. Never­theless, evident formation of colonies which might be successively cultivable has never been accomplished.

With regard to relation of these isolated strains and M. leprae, it was reported by the Technical Committee on Bacteriology, the Seventh International Leprosy Con­gress, 1958, that the various mycobacteria isolated theretofore from lepromatous tis-

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41, 3 Yoshie: Microbiology of M.leprae 367

sues were all incidental or passenger strains and none could be regarded as the causa­tive agent of leprosy. Furthermore, ac­cording to a presentation made by Kirch­heimer and Prabhakaran at the Ninth Inter­national Leprosy Congress, 1968, a series of 15 strains listed under Mycobacterial "Spe­cies" in the "Catalogue of Cultures," 7th edition, 1964, of the American Type Cul­ture Collection (ATCC) , as believed at one time to be leprosy bacillus, and some mycobacteria listed in the catalogue as iso­lated originally from ticks experimentally infected with lepromatous material, were examined for dopa-oxidation activity char­acteristic of M. leprae and by mouse foot pad tests. The results showed that all the 15 isolates examined had little resemblance to M. leprae (18).

Studies on the cultivation of M. leprae were carried forth actively during the peri­od of about 50 years after Hansen's discov­ery of the leprosy bacillus, yielding a num­ber of reports on isolates from leprous ma­terials. The study along this line, however, remained temporarily in a state of extreme depression under the influence of World War II. On the occasion of the Seventh International Leprosy Congress, the Com­mittee on Bacteriology and Pathology presented the following two objectives as the basic approach to the cultivation of M. leprae to be achieved in the future : first, to conduct biochemical studies on deficits and impediments of the enzyme system of non­cultivated mycobacteria; and, second, to look for tissue cell systems or other biologic systems which may p ermit substitution for the natural hosts.

Meanwhile, bacteriologic studies of the biologic characteristics of microorganisms, especially biochemical physiology, were ac­tively pursued after World War II with consequent rapid progress in the under­standing of the basic physiologic aspects of bacteria such as structure, mechanism of respiration, nutrition and genetics. Thanks to the advances in general microbiology, a rational way of approach to the microbiolo­gy of M. leprae, especially to its cultiva­tion, has emerged on the basis of the newly gained physiologic knowledge and new concepts. At the symposium on research in

leprosy held at Baltimore in 1961, at the EightJl International Leprosy Congress, and at the LWM-AFIP Conference on re­search problems in leprosy, held at Wash­ington in 1965, the necessity of assessment of the problem of cultivation of M. leprae and M. lepraemurium from a new point of view on the basis of new microbiologic knowl.edge was generally admitted to be imperative.

Tissue culture. Tissue culture of small pieces of lepromatous nodule was first tried about 20 years following the introduction of the tissue culture method by Harrison ( 1907) and Carrel (1910). With the subse­quent immense progress in tissue culture technics, numerous human and mammalian cell strains were isolated and maintained and, by utilizing them, active investiga­tions were carried out on cultivation of M. leprae and M. lepraemurium. Garbutt et al (n) reported multiplication of rat leprosy bacilli in culhues of rat fibroblasts. Chang (l\) achieved successful cultivation of M. lepraemurium for 20 to no days within mouse peritoneal macrophages maintained in vitro, thereby demonstrating a definite intracellular multiplication of the bacilli as­sociated with elongation.

Outweighing any other consideration in the cultivation of M. leprae in cell cultures is the search for a host cell system suitable for parasitization by this organism. Several types of cultures have been employed. Among these have been : 1) explant cul­tures made from humall tissues; 2) cell lines of animal origin such as L cells, rat fibroblasts and monkey kidney cells; 3) cell lines of human origin such as KB, HeLa, amnion, embryonic lung, etc. ; 4) cell strains derived from human tissues, chiefly normal skin fibroblasts, fibroblasts from lep­romatous skin and fetal spinal ganglion fibroblasts; and 5) mouse peritoneal macro­phages.

Although extensive work has been done along these lines, very little success has been achieved. Bacillary elongation associ­ated with a limited degree of multiplication was observed in some culture systems, as for example, in Schwannoma cells, fibro­blasts derived from lepromatous skin and in mouse macrophages .

368 lnternatiorwt Journal of Leprosy 1973

Biochemical studies. Biochemists having special interests in bacteriology have be­come more and more involved in the prob­lem of bacterial growth and, as a result, there have been significant contributions both theoretical and practical. It has be­come clear that the particular culture media which bacteriologists use for cultiva­tion of pathogenic bacteria which are oth­erwise difficult to cultivate require supple­ments of substances which the parasites under study are incapable of synthesizing for their own- growth requirements. A sub­stantially marked advance has thus been made in the knowledge of microbiology in the last 40 years. With this newly acquired knowledge and advanced technics, the study of cultivation of M. leprae has under­gone a drastic change, both with respect to theoretical and practical aspects.

In the study of the cultivation of M. leprae in cell-free media, it has been an object of prime importance in the past 20 years to investigate the biochemical system involved in the supply of energy required by the leprosy bacillus for growth in vitro. Investigations of culture media, cofactor and growth faotor supplements, and envi­ronmental conditions for cultivation of the leprosy bacillus have been increasingly ini­tiated on the basis of biochemical under­standing of the metabolic capabilities and deficiencies of the organism. The pressing practical necessity arose for an ample sup­ply of fresh bacilli at any given time, and definite amounts of pure bacilli uncontami­nated with tissue or tissue debris were needed to facilitate bacteriologic and bio­chemical studies of M. leprae. These, not being generally available, studies with M. lepraemurium as the model system for M. leprae came to be performed in many laboratories.

Experimentation on oxygen consumption by M. lepraemurium by the manometric method of Warburg with the various sub­stances that have been used for the cultiva­tion of mycobacteria revealed that the or­ganism is destitute of the capacity to utilize any of the substances usually employed as a source of energy (11). Subsequently, evi­dence has been obtained of a correlation between mycobacterial oxidative metabo-

\ism and the ease with which they can be cultivated in vitro (12).

In recent years isotope tracer technics became available for the study of bacterial metabolism and it became practicable to detect and measure the extremely minute amounts of radioactive substrates oxidized by or incorporated into bacteria. These investigations by isotope tracer technics demonstrated M. lepraemurium capability in oxidizing and assimilating exogenous substrates, and that the rate for the utiliza­tion of the exogenous substrates is remark­ably low, being lower even than that shown by noncultivated Rickettsiae and Chla­mydiae (35). Recently, studies of in vitro fatty acid uptake by M. lepraemurium have been conducted by the use of 14C_ labeled fatty acids. Incorporations of radi­oactivity from 14C-decanoate into M. lep­raemurium were observed, and these in­corporations were demonstrated to be due to a biological function of this organism (19) .

Energy yielding p.athways of mycobac­teria depend mainly on respiratory systems because of their absence of anaerobic glycol­ysis. The non cultivable state of leprosy bacilli seems to depend on minimal capaci­ty for respiration. It has been observed that M. lepraemurium separated from infected tissues does not exhibit detectable cyto­chrome adsorption bands which are readily ascertainable in culturable mycobacteria. This seems to indicate that leprosy bacilli have genetically lost the adaptive capacity of a respiratory system. If a clearer under­standing of the defect in respiratory sys­tems of mycobacteria can be obtained it may be possible to find some approach to the puzzling problem of cultivating leprosy bacilli.

It has been found that as the difficulty in cultivation in vitro increases from saprophytic to attenuated and finally to parasitic strains of mycobacteria, their res­piratory activities diminish sharply. Both M. lepraemurium cells obtained from lep­rous nodules of infected mice and in vivo grown BCG cells exhibit extremely faint activity or are even devoid of enzymes of the cytochrome series. Of pmticular note is the finding that in vitro cultivated BCG

41, 3 Yoshie: Microbiology of M.leprae 369

cells bccome cytoohrome deficient, lik~ M. lepraemurium, when grown in vivo ( ~O ). Clarification of the mechanism whereby BCG cells lose the ability to synthesize cytochromcs may throw light on the basic biochemical features of leprosy bacilli.

M. lepraemurium has been shown to be incapable of initiating the TCA cycle; hcnce, an incomplete TCA cycle enzyme system exis·ts in this bacillus (23 ). Experi­mcntal evidencc has also been secured for the pattern of TCA cycle impediment seen with strongly respiratory-impaired mutants of M. smegmatis which resemble that of the defect in TCA cycle observed with M. lepraem'Urium (21).

It is reasonable to think that an under­standing of the changes in the carbon, nitrogen and oxidative metabolism that must occur when mycobacteria undergo successful transference would provide a logical basis for analyzing the palticular steps which host-dependent organisms are unable to accomplish (24).

Cultivation trials in cell-free media. Extensive studies on the cultivation of M. leprae in cell -free media have been under­taken in many laboratories throughout the world. Successful growth has been claimed by several groups of investigators during the last decade. Howcver, none of these are as yet accepted as successful growth . Most of the isolated organisms have been iden­tificd as strains of saprophytic mycobacteria or Runyon group III of atypical mycobac­teria and, with some organisms, no suffi­cient identification tests for M. leprae have been performed.

With respect to cultivation trials with M. lepraemurium, on the other hand, there are a few recent reports of considerable inter­est.

Formation of slow growing, dull yellow, rough colonies was evident following a heavy inoculation of M. lepraemurium onto cgg yolk medium and 16 serial subcultiva­tions have been successful. The rate of positive serial subcultiv'ation has been 46/ 469 with the Hawaiian strain and 25/ 63 with thc · Keishicho stra;n, respectively. Thesc bacilli wcre demonstrated to pro­duce characteristic murine leprosy in mice (27) . .

A growth of M . lepraemurium could b e scen in the cell-free medium, NC-5 medi­um, containing a-ketoglutaric acid, cyto­chrome C, hemin, I-cysteine, and goat serum in Kirchner medium. The b acilli gradually elongated and began to multiply approximatcly t en days after incubation. Thereafter, the number of bacilli increased and rcachcd about 50 times the inoculum size in a period of 60 days. These bacilli maintained their pathogenicity for mice and produccd charactcristic murine leprosy (~5) .

Although an attempt at cultivation of M. leprae was madc under the same condition as cmployed in M. lepraemurim, the results obtained showed no morphological changes in the bacilli and no demonstration of mul­tiplicati on by M. leprae.

Advances in identification of M. leprae. The WHO Expelt Committee on Leprosy, 1970, discussed the identification of M. leprae and statcd' : "In all cultivation work it is important to prove the viability of the purported growth and to identify it by the methods now available (the inoculation of mouse foot pads, lepromin t ests, enzymatic studies of DOPA oxidation, and serological identification of leprosy nodular extract an­tigen )."

These points may be elaborated to indi­cate in greater detail present data available for thc identificati on of M. leprae. Charac­teristic lesions, particularly involvement of peripheral nerves, should be found on inoc­ulation into normal or thymectomized­irradiated mice. The skin reactivity elicited in lepromatous and tuberculoid patients by antigen prepared from the culture in ques­tion, should conform with their reaction to lepromin. Assay of the bacteria for DOPA oxidase activity is specific to M. leprae. It has becn recently demonstrated that M. leprae, unlike other species of mycobac­teria, can oxidize 3,4-dihydroxyphenyl­alaninc ( DOPA ) and that the DOPA ox­idasc of M. leprae differs from that present in mammalian ti ssucs (28). Additionally, it has rccently been shown by immunodifus­sion and immunofluorescence studies that M. leprae contains a protein antigen showing an cntirely different specificity from other species of mycobacteria. It was

370 International] ournal of Leprosy 1973

found that leprosy nodule extact ( NE ) contained at least two antigens which are easily differentiated ftom human serum proteins by immunodiffusion. One of these antigens is a heat stable polysaccharide, and the other is a heat labile protein which gave a single precipitation line with anti­NE serum absorbed with human serum. Insomuch as this antigen is highly specific for M. leprae, serological identification of M. leprae became practicable through the use of antiserum prepared against the anti­gen (1).

Lepromin reaction. The Mitsuda react.ion described by Mitsuda in 1919 (22), and Hayashi in 1933 (14), was interpreted as expressing the resistance of individual hosts to the leprosy bacillus, by the Immunology Committee of the Sixth International Lep­rosy Congress in 1953. Standards for the preparation of lepromin and criteria for evaluation of the result of the test were stipulated. Investigations were carried out over the ensuing years to examine the relationship between Mitsuda reactivity and the resistance to leprosy and, under the auspices of WHO, standardization of bacillary concentrations in lepromin was investigated. An antigen containing 160 million bacilli per milliliter was initially recommended as the standard by the Eighth International Congress of Leprology and the Third WHO Expert Committee on Leprosy.

Subsequent studies of the preservation of lepromin showed that the bacterial cotmt of lepromin preserved in a refrigerator is re­duced to about 74% of the original bacterial count after three years and about 70% after five years, while the lepromins preserved by lyophilization showed no decrease in their bacterial count. Lepromin can be preserved for three years in a refrigerator without appreciable loss of potency. Ly­ophilization of lepromin should be recom­mended for a prolonged preservation. Studies in recent years have been pursued with diluted lepromin in attempts to deter­mine more specifically what bacterial con­centration of lepromin is required for max­imum potency and efficacy of the lepromin test.

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